Bhawani et al Tropical Journal of Pharmaceutical Research June 2010; 9 (3): 301-313 © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved . Available online at http://www.tjpr.org Review Article Thin-Layer Chromatographic Analysis of Steroids: A Review SA Bhawani* 1, O Sulaiman 1, R Hashim 1 and MN Mohamad Ibrahim 2 1Division of Bio-Resource, Paper and Coatings Technology, School of Industrial Technology, 2School of Chemical Sciences, Universiti Sains Malaysia, 11800, Pulau Pinang, Malaysia Abstract Thin layer chromatography has been used for the analysis of natural and synthetic steroids in various environmental materials. This review focuses mainly on steroid analysis in environmental materials such as pharmaceuticals, plant products and other biological specimens. The most widely investigated biological specimens are urine and blood plasma or serum. Various chromatographic systems useful for the identification; separation and quantification of surfactants are also reported in this review. Keywords: Steroids; Thin layer chromatography; Environmental materials; Biological specimens Received: 15 November 2009 Revised accepted: 18 March 2010 *Corresponding author: E-mail: [email protected] Trop J Pharm Res, June 2010; 9 (3): 301 Bhawani et al INTRODUCTION androgens. Steroids, such as nandrolone, dromostanolone, stanozolol, are often used Steroids are terpenoid lipids characterized by illegally to increase the performance of the sterane or steroid nucleus: a carbon competitive athletes of almost all age groups. skeleton with four fused rings, generally They are banned in most sports competitions arranged in a 6-6-6-5 fashion. Steroids vary such as the Olympic Games. by the functional groups attached to these rings and the oxidation state of the rings. The Classification of Steroids specificity of their different biological actions is due to the various groups attached to a Steroids have been classified into a number common nucleus. When alcohol groups (OH) of groups by Scott [1] based on their are attached, steroids should properly be functions as follows: (i) sterols and steroid called sterols (e.g., cortisol), whereas ketone alcohols, usually with double bonds; (ii) sex groups (C=O) make them sterones (e.g., hormones - steroids produced mainly in the aldosterone). testis (androgens) or ovary (estrogens); (iii) adrenocortical hormones - steroids produced Steroids comprise a large group of in the cortex of the adrenal gland; (iv) bile substances that mediate a very varied set of acids - steroids usually bonded to taurine or biological responses. The most widespread in glycine and functioning as emulsion- the body is cholesterol, an essential stabilizing agents in the intestine; (v) component of cell membranes and the sapogenins - plant products with a steroid starting point for the synthesis of other bonded to carbohydrates; (vi) cardiac steroids - sex hormones, adrenal cortical glycosides - plant products similar to hormones, and the bile salts. Steroids (e..g., sapogenins and used as heart stimulants; glucocorticoids, mineralocorticoids, and (vii) vitamin D androgens,estrogens and progestagens) have major responsibilities as hormones, ANALYSIS OF STEROIDS controlling metabolism, salt balance, and the development and function of the sexual Many procedures used for the quality control organs as well as other biological differences and quality assurance of steroids are based between the sexes. Steroids in the form of on classical methods of analysis. However, bile salts (e.g., salts of cholic and deoxycholic the need for improved precision and accuracy acid and their glycine and taurine conjugates) has led to the increased use of instrumental assist in digestive processes, while another analysis. Thus, the development of fast and steroid is a vitamin (calcitriol) that takes part reliable analytical methods for quality control, in calcium control. Steroids (naturally including the identification of synthesis by- occurring or synthetic) such as products and purity tests, are both important methylprednisolone, hydrocortisone, gluco- and challenging. Thin-layer chromatography cortisteroids, corticosteroids, squalamine, (TLC) continues to be an important method oestrogens, androgens, are also used for the for qualitative analysis of steroids because of treatment of various diseases such as allergic its inherent advantages - many samples can reactions, arthritis, some malignancies, and be analyzed simultaneously and quickly, and diseases resulting from hormone deficiencies multiple separation techniques and detection or abnormal production. In addition, synthetic procedures can be applied. This review steroids (e.g., mifepristone) that mimic the presents the contribution of thin-layer action of progesterone are widely used as chromatography in the analysis of steroids oral contraceptive agents. Other synthetic from 1990-2009. It addresses most aspects steroids (e.g., oxandrolone) are designed to of thin-layer chromatography, including mimic the stimulation of protein synthesis and detection, separation and quantification of muscle-building action of naturally occurring steroids. Trop J Pharm Res, June 2010; 9 (3): 302 Bhawani et al Thin-layer chromatography of steroids TLC continues to be an important method for the determination of steroids because of its Szepesi and Gazdag wrote a book chapter advantages. Many samples can be analyzed on the TLC of steroids, and it included simultaneously and quickly at relatively low information on sample preparation as well as cost; also, multiple separation techniques and stationary-phase and mobile-phase systems detection procedures can be applied and the useful for the separation of steroidal detection limits are often in the low nanogram pharmaceuticals [2]. The authors also range, and quantitative densitometric provided detailed methods of detection and methods are accurate. Modern approaches in quantification of steroids, and later on, thin-layer chromatography enable analysts to updated their review to include coverage separate and determine steroids in complex through 1994 [3]. Dreassi et al [4] have also mixtures, including various environmental reviewed the application of TLC to steroids in samples. Steroids and their metabolites are pharmaceutical analysis while Jain has analyzed by thin-layer chromatography in a provided some information on the analysis of variety of samples such as biological steroid hormones in his review on TLC in samples, plants and pharmaceutical clinical chemistry [5]. formulations. Table 1(a)-(e) shows several thin-layer chromatographic systems designed for the analysis of steroids [6-29]. Table 1(a): Thin-layer chromatographic analysis of steroids Analyte Stationar Mobile phase Remarks Ref y phase Cholestrol, RP- Acetronitrile/methanol, Investigation of the retention behavior 6 allylestrenol, HPTLC acetronitrile/water and of 12 sreroids.Mixture of 10g copper pregnanediol, plates methanol/water in different binary sufate and 5 mL o-phosphoric acid etc. mixtures (86%) dissolved in 95 mL methanol. Androgens Silica Cyclohaxane/ethylacetate/ethanol HPTLC separation of anabolic and gestagens (24:16:1) and Androgens. chloroform/benzene/ethanol(36:4:1 Detected by fluorescence after ) in one direction immersion in a 5% sulfuric acid-ethanol ;chloroform/acetone (9:1) and solution for 30 sec and viewed under hexane/dichloromethane/ UV366nm. acetronitrile (4:3:2) in second direction for androgens and gestagens respectively Steroids Silica Chloroform/ethanol/water Detection under 8 (188:12:1) UV.Quantification by radioimmunoassay. Progesterone, Silica Methanol/ethylacetate/chloroform/ Programmed multiple development of 9 testosterone, methylenechloride (first inverse analysis of steroids. testosterone gradient program) and Detected under UV 254.Densitometry hydrogen methanol/chloroform (second was used for the quantification. sulfate sodium inverse gradient program) salt, etc. Oxo-steroids Silica gel Chloroform/methanol (97:3) Measurement of 17-oxo steroids in biol. 10 F254 Fluids with TLC and fluorometric scanning detection. Dansylhydrazine was used as a prelabeling reagent. Linearity of fluorescence detection was obtained at 30-1000 ng. Trop J Pharm Res, June 2010; 9 (3): 303 Bhawani et al Table 1(b): Thin-layer chromatographic analysis of steroids ( contd. ) Analyte Stationary Mobile phase Ref phase Hydrocortisone, Benzene/ Use of colour 11 prednisolone, ethylacetate (1:1) photodocumentation of UV- mesylate, etc. irradiated thin-layer chromatograms for the analysis of steroids.Detected by spraying with a 10% ethanolic solution of sulfuric acid followed by heating at 100 oC for 2 to 4 min Anabolic steroids Silica Chloroform/aceto Analysis of anabolic steroids by 12 ne (9:1) in one high-performance thin-layer direction and chromatography.Detected by cyclohexane/ethy spraying with 10% sulfuric acid in lacetate/methano methanol and heating for 10 min at l (117:78:11) in 95 oC in day light and under UV the opposite 366nm.Further confirmation was direction done by GC-MS Cortisone, NH 2 F 245s Chloroform/ethan Analysis and separation of steroids 13 hydrocortisone, ol/formic acid on NH 2 layers. estradiol, (50:10:10), Estradiol benzoate, Chloroform/meth estriol,estrone, anol (95:5), methyltestosterone, Chloroform/1- testosterone, propanol/formica testosterone cid (50:10:5) propionate, prednisolone, pegnandiol and triol, progeterone and Reichstein’s S Cortisole, cortisone, NH 2 F 245s Chloroform/ethan Separation